Enhancing the circulating half-life of antibody-based fusion proteins

ABSTRACT

Disclosed are compositions and methods for enhancing the circulating half-life of antibody-based fusion proteins. Disclosed methods and compositions rely on altering the amino acid sequence of the junction region between the antibody moiety and the fused protein moiety in an antibody-based fusion protein. An antibody-based fusion protein with an altered amino acid sequence in the junction region has a greater circulating half-life when administered to a mammal. Disclosed methods and compositions are particularly useful for reducing tumor size and metastasis in a mammal.

RELATED APPLICATIONS

This application claims priority to, and the benefit of U.S. Ser. No.60/181,768, filed Feb. 11, 2000, the disclosure of which is incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention relates generally to fusion proteins. Morespecifically, the present invention relates to methods of enhancing thecirculating half-life of antibody-based fusion proteins.

BACKGROUND OF THE INVENTION

The use of antibodies for treating human diseases is well establishedand has become more sophisticated with the introduction of geneticengineering. Several techniques have been developed to improve theutility of antibodies. These include: (1) the generation of monoclonalantibodies by cell fusion to create “hybridomas”, or by molecularcloning of antibody heavy (H) and light (L) chains fromantibody-producing cells; (2) the conjugation of other molecules toantibodies to deliver them to preferred sites in vivo, e.g.,radioisotopes, toxic drugs, protein toxins, and cytokines; (3) themanipulation of antibody effector functions to enhance or diminishbiological activity; (4) the joining of other proteins such as toxinsand cytokines with antibodies at the genetic level to produceantibody-based fusion proteins; and (5) the joining of one or more setsof antibody combining regions at the genetic level to producebi-specific antibodies.

Proteins can be joined together through either chemical or geneticmanipulation using methods known in the art. See, for example, Gillieset al., Proc. Natl. Acad. Sci. USA 89:1428-1432 (1992); and U.S. Pat.No. 5,650,150.

However, the utility of recombinantly-produced antibody-based fusionproteins may be limited by their rapid in vivo clearance from thecirculation. Antibody-cytokine fusion proteins, for example, have beenshown to have a significantly lower in vivo circulating half-life thanthe free antibody. When testing a variety of antibody-cytokine fusionproteins, Gillies et al. reported that all of the fusion proteins testedhad an α phase (distribution phase) half-life of less than 1.5 hours.Indeed, most of the antibody-based fusion proteins were cleared to 10%of the serum concentration of the free antibody by two hours. See,Gillies et al., BIOCONJ. CHEM. 4: 230-235 (1993). More recently, it wasshown that antibody-based fusion proteins with reduced binding affinityfor an Fc receptor have enhanced circulating half-lives. It was alsoshown that a reduced binding affinity for the Fc receptor interferedwith some of the antibody effector functions such as antibody-dependentcellular cytotoxicity (ADCC), but did not interfere with other functionssuch as complement fixation or antigen binding. See Gillies at al.,Cancer Res. 59(9):2159-66 (1999).

In some cases, such as the treatment of cancer or viral diseases, itwould be desirable to maintain antibody effector functions and longcirculating half-life. Therefore, there is a need in the art foradditional methods of enhancing the in vivo circulating half-life ofantibody-based fusion proteins.

SUMMARY OF THE INVENTION

Immunoglobulin G (IgG) molecules interact with multiple classes ofcellular receptors including three classes of Fcγ receptors (FcγR)specific for the IgG class of antibody, namely FcγRI, FcγRII andFcγRIII. They also interact with the FcRp class of receptor in apH-dependent manner with little or no binding at neutral pH but highbinding at a pH of 6.0.

The serum half-life of an antibody is influenced by the ability of thatantibody to bind to an Fc receptor (FcR) and to the Fc protectionreceptor (FcRp). The serum half-life of immunoglobulin fusion proteinsis also influenced, for example, by the ability to bind to suchreceptors (Gillies et al., Cancer Res. 59:2159-66 (1999)).

The invention discloses the surprising observation that, within fusionproteins comprising an immunoglobulin (Ig) moiety and anon-immunoglobulin (non-Ig) moiety, alteration of amino acids near thejunction of the two moieties dramatically increases the serum half-lifeof the fusion protein. The observation is surprising because the aminoacid changes affect protein surfaces that are distinct from theinteraction surfaces of the Fc region with the Fc receptors and with theFc protection receptor. In addition, the amino acid changes of theinvention have their effect even when the known Fc receptor and Fcprotection receptor are not primarily determining the serum half-life ofthe fusion protein. Thus, the amino acid alterations of the inventioncan be combined with amino acid alterations affecting the interactionwith Fc receptor and/or Fc protection receptor to achieve synergisticeffects.

The present invention provides fusion proteins containing animmunoglobulin in which the serum half-life is improved as a result ofalterations that are at sites distinct from the Fc region's interactionsurface with Fc receptor and Fc protection receptor (FcRp). The presentinvention also provides methods for the production of fusion proteinsbetween an immunoglobulin moiety and a second, non-immunoglobulinprotein having an improved serum half-life.

The alterations in the amino acid sequence of the fusion protein arepreferentially at the junction of the Ig moiety and the non-Ig moiety.The junction region of the fusion protein contains alterations that,relative to the naturally occurring sequences of the Ig heavy chain andnon-Ig protein, preferably lie within about 10 amino acids of thejunction point. More preferably, the amino acid changes cause anincrease in hydrophobicity. Even more preferably, the amino acid changesinvolve changing the C-terminal lysine of the antibody moiety to ahydrophobic amino acid such as alanine or leucine. In a preferredembodiment, the fusion protein of the invention comprises an Ig heavychain, preferably located N-terminal to a second, non-Ig protein.

In another embodiment of the invention, the binding affinity of fusionproteins for FcRp is optimized by alteration of the interaction surfaceof the Fc moiety that contacts FcRp. The important sequences for thebinding of IgG to the FcRp receptor have been reported to be located inthe CH2 and CH3 domains. According to the invention, alterations of thefusion junction in a fusion protein are combined with alterations ofFc's interaction surface with FcRp to produce a synergistic effect. Insome cases it may be useful to increase the interaction of the Fc moietywith FcRp at pH 6, and it may also be useful to decrease the interactionof the Fc moiety with FcRp at pH 8. Such modifications includealterations of residues necessary for contacting Fc receptors oraltering others that affect the contacts between other heavy chainresidues and the FcRp receptor through induced conformational changes.Thus, in a preferred embodiment, an antibody-based fusion protein withenhanced in vivo circulating half-life is obtained by first linking thecoding sequences of an Ig constant region and a second,non-immunoglobulin protein and then introducing a mutation (such as apoint mutation, a deletion, an insertion, or a genetic rearrangement) inan IgG constant region at or near one or more amino acid selected fromIle 253, His 310 and His 435. The resulting antibody-based fusionproteins have a longer in vivo circulating half-life than the unmodifiedfusion proteins.

In certain circumstances it is useful to mutate certain effectorfunctions of the Fc moiety. For example, complement fixation may beeliminated. Alternatively or in addition, in another set of embodimentsthe Ig component of the fusion protein has at least a portion of theconstant region of an IgG that has reduced binding affinity for at leastone of FcγRI, FcγRII or FcγRIII. For example, the gamma4 chain of IgGmay be used instead of gamma 1. The alteration has the advantage thatthe gamma4 chain results in a longer serum half-life, functioningsynergistically with one or more mutations at the fusion junction.Accordingly, IgG2 may also be used instead of IgG1. In an alternativeembodiment of the invention, a fusion protein includes a mutant IgG1constant region, for example an IgG1 constant region having one or moremutations or deletions of Leu₂₃₄, Leu₂₃₅, Gly₂₃₆, Gly₂₃₇, Asn₂₉₇, orPro₃₃₁. In a further embodiment of the invention, a fusion proteinincludes a mutant IgG3 constant region, for example an IgG3 constantregion having one or more mutations or deletions of Leu₂₈₁, Leu₂₈₂,Gly₂₈₃, Gly₂₈₄, Asn₃₄₄, or Pro₃₇₈. However, for some applications, itmay be useful to retain the effector function that accompanies Fcreceptor binding, such as ADCC.

In a preferred embodiment, the second, non-immunoglobulin moiety of thefusion protein is a cytokine. The term “cytokine” is used herein todescribe naturally occurring or recombinant proteins, analogs thereof,and fragments thereof which elicit a specific biological response in acell which has a receptor for that cytokine. Preferably, cytokines areproteins that may be produced and excreted by a cell. Cytokinespreferably include interleukins such as interleukin-2 (IL-2), IL-4,IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18,hematopoietic factors such as granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF) anderythropoietin, tumor necrosis factors (TNF) such as TNFα, lymphokinessuch as lymphotoxin, regulators of metabolic processes such as leptin,interferons such as interferon α, interferon β, and interferon γ, andchemokines. Preferably, the antibody-cytokine fusion protein of thepresent invention displays cytokine biological activity.

In an alternative preferred embodiment, the second, non-immunoglobulinmoiety of the fusion protein is a ligand-binding protein with biologicalactivity. Such ligand-binding proteins may, for example, (1) blockreceptor-ligand interactions at the cell surface; or (2) neutralize thebiological activity of a molecule (e.g., a cytokine) in the fluid phaseof the blood, thereby preventing it from reaching its cellular target.Preferably, ligand-binding proteins include CD4, CTLA-4, TNF receptors,or interleukin receptors such as the IL-1 and IL-4 receptors.Preferably, the antibody-receptor fusion protein of the presentinvention displays the biological activity of the ligand-bindingprotein.

In yet another alternative preferred embodiment, the second,non-immunoglobulin moiety of the fusion protein is a protein toxin.Preferably, the antibody-toxin fusion protein of the present inventiondisplays the toxic activity of the protein toxin.

In yet other preferred embodiments, the second, non-immunoglobulinmoiety of the fusion protein is a hormone, neurotrophin, body-weightregulator, serum protein, clotting factor, protease; extracellularmatrix component, angiogenic factor, anti-angiogenic factor, or anothersecreted protein or secreted domain. For example, CD26, IgE receptor,polymeric IgA receptor, other antibody receptors, Factor VIII, FactorIX, Factor X, TrkA, PSA, PSMA, Flt-3 Ligand, endostatin, angiostatin,and domains of these proteins.

In yet other embodiments, the second, non-immunoglobulin moiety is anon-human or non-mammalian protein. For example, HIV gp120, HIV Tat,surface proteins of other viruses such as adenovirus, and RSV, other HIVcomponents, parasitic surface proteins such as malarial antigens, andbacterial surface proteins are preferred. These non-human proteins maybe used, for example, as antigens, or because they have usefulactivities. For example, the second, non-immunoglobulin moiety may bestreptokinase, staphylokinase, urokinase, tissue plasminogen activator,or other proteins with useful enzymatic activities.

According to the invention, the non-immunoglobulin moiety can be aportion of a protein. Preferably, the non-Ig protein moiety is a proteinportion that substantially retains the functional and or structuralproperties of an intact protein. In a preferred embodiment, the non-Igprotein moiety is a functional or structural portion of a proteindescribed herein.

In a preferred embodiment, the antibody-based fusion protein comprises avariable region specific for a target antigen as well as a constantregion, either of which is linked through a peptide bond to a second,non-immunoglobulin protein. The constant region may be the constantregion normally associated with the variable region, or a different one,e.g., variable and constant regions from different species. The heavychain may include any combination of one or more CH1, CH2, or CH3domains. Preferably, the heavy chain includes CH1, CH2, and CH3 domains,and more preferably, only CH2 and CH3 domains. In one embodiment, theantibody-based one fusion protein comprises an Fv region with fusedheavy and light chain variable regions. Also embraced within the term“fusion protein” are constructs having a binding domain comprisingframework regions and variable regions (i.e., complementaritydetermining regions) from different species, such as are disclosed byWinter, et al., Great Britain Patent No. 2,188,638. Antibody-basedfusion proteins comprising a variable region preferably displayantigen-binding specificity. In yet another preferred embodiment, theantibody-based fusion protein further comprises a light chain. Theinvention thus provides fusion proteins in which the antigen-bindingspecificity and activity of an antibody are combined with the potentbiological activity of a second, non-immunoglobulin protein, such as acytokine. A fusion protein of the present invention can be used todeliver selectively the second, non-immunoglobulin protein to a targetcell in vivo so that the second, non-immunoglobulin protein can exert alocalized biological effect.

In an alternative preferred embodiment, the antibody-based fusionprotein comprises a heavy chain constant region linked through a peptidebond to a second, non-immunoglobulin protein, but does not comprise aheavy chain variable region. The invention thus further provides fusionproteins which retain the potent biological activity of a second,non-immunoglobulin protein, but which lack the antigen-bindingspecificity and activity of an antibody.

In preferred embodiments, the fusion protein comprises two chimericchains comprising at least a portion of a heavy chain and a second,non-Ig protein linked by a disulfide bond.

In preferred embodiments, the fusion proteins of the invention areuseful to treat cancer, viral infections, immune disorders, and toenhance the growth (including proliferation) of specific cell types.

The invention also features DNA constructs encoding the above-describedfusion proteins, and cell lines, e.g., myelomas, transfected with theseconstructs.

These and other objects, along with advantages and features of theinvention disclosed herein, will be made more apparent from thedescription, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pharmacokinetic behavior of the KS-IL-2 fusion proteinand various mutant fusion proteins containing substitutions of theantibody heavy chain's C-terminal lysine moiety or other alterationsdescribed in the Examples. Levels of antibody or fusion protein weremeasured by an ELISA that tests for IL-2 (FIG. 1A) or human Fc (FIG.1B).

FIG. 2 shows the pharmacokinetic properties of KS-IL-2 fusion proteinscarrying either the gamma1 or gamma4 chain with either the wild-typelysine or the lysine-to-alanine mutation at the C-terminus of theantibody heavy chain. Levels of antibody or fusion protein were measuredby an ELISA that tests for the IL-2 moiety.

FIG. 3 shows the pharmacokinetic properties of fusions of a humanantibody to Tumor Necrosis Factor alpha (TNFalpha). Levels of fusionprotein were measured by an ELISA that tests for the human Fc region.Shown are the levels of an intact antibody-TNFalpha fusion protein(black diamonds) and the levels of an otherwise identical fusion proteinin which the C-terminal lysine of the antibody moiety has been deleted(gray squares).

FIG. 4 shows the binding of antibody-IL-2 fusion proteins to membranesof fixed J774 cells, which are rich in the FcγR class of receptor. Shownare the binding of a non-mutant KS-IL-12 fusion protein (black diamonds)and a KS-IL-12 fusion protein carrying a mutation of the heavy chainC-terminal Lysine to Alanine (gray squares).

FIG. 5 shows the effect of antibody-cytokine fusion protein treatment ofBalb/C mice bearing subcutaneous tumors derived from CT26 coloncarcinoma cells that were engineered to express human EpCAM, the antigenfor KS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibody fusion proteins having one ormore mutations at the junction between the Ig and non-Ig moieties whichincrease the circulating half lives of the fusion proteins. The mutantfusion proteins of the invention have the advantageous property thattheir serum half-life is improved without affecting the interaction ofthe antibody moiety with either of the two knownpharmacokinetic-determining receptors in the body: Fc receptor and FcRp.

In general, an antibody-based fusion protein of the invention comprisesa portion of an immunoglobulin (Ig) protein joined to anon-immunoglobulin (non-Ig) protein, such that the amino acid sequenceof the region spanning the junction between the Ig and non-Ig proteinshas at least one mutation when compared to the wild-type amino acidsequences of the Ig and non-Ig proteins.

In one embodiment, at least one mutation is in the C-terminal region ofthe Ig portion. In another embodiment, at least one mutation is in theN-terminal region of the non-Ig protein. In a further embodiment, thefusion protein contains at least one mutation in the C-terminal regionof the Ig portion, and at least one mutation in the N-terminal region ofthe non-Ig protein. A mutation may be a point mutation, an insertion, adeletion, or a gene rearrangement. In preferred embodiments the mutationincreases the hydrophobicity of the junction region. For example, themutation replaces a charged or ionizable amino acid with a non-chargedor hydrophobic amino acid (e.g., a Lys, Arg or other ionizable residueis replaced with an Ala, Leu, Gly, Trp or other non-charged orhydrophobic residue).

In an optional embodiment, a spacer or linker peptide is insertedbetween the Ig and non-Ig proteins. The spacer or linker peptide ispreferably non-charged, more preferably non-polar, and or hydrophobic.The length of a spacer or linker peptide is preferably between 1 andabout 100 amino acids, more preferably between 1 and about 50 aminoacids, or between 1 and about 25 amino acids, and even more preferablybetween 1 and about 15 amino acids. In another embodiment of theinvention, the Ig and non-Ig moieties of the fusion protein are joinedvia a spacer or linker peptide, and there is at least one mutation ineither one or both of the Ig and non-Ig moieties. In an alternativeembodiment of the invention, the Ig and non-Ig moieties are separated bya synthetic spacer, for example a PNA spacer, that is preferablynon-charged, more preferably non-polar, and or hydrophobic.

According to the invention, an immunoglobulin (Ig) chain is animmunoglobulin protein or a portion of an immunoglobulin protein thatincludes a variable or a constant domain. An Ig chain is preferably aportion of an immunoglobulin heavy chain, for example, an immunoglobulinvariable region capable of binding a preselected cell-type. In apreferred embodiment, the Ig chain comprises a variable region specificfor a target antigen as well as a constant region. The constant regionmay be the constant region normally associated with the variable region,or a different one, e.g., variable and constant regions from differentspecies. In a more preferred embodiment, an Ig chain includes a heavychain. The heavy chain may include any combination of one or more CH1,CH2, or CH3 domains. Preferably, the heavy chain includes CH1, CH2, andCH3 domains, and more preferably only CH2 and CH3 domains. In oneembodiment, the portion of the immunoglobulin includes an Fv region withfused heavy and light chain variable regions.

According to the invention, a non-immunoglobulin protein includes anaturally occurring protein that is not an immunoglobulin, or asynthetic or recombinant protein that is not an immunoglobulin, or afragment of any of the above. In a preferred embodiment, anon-immunoglobulin protein includes a functional domain such as a ligandbinding domain, an enzymatic domain, a regulatory domain, or a domainthat interacts with one or more cellular factors. In an alternativeembodiment, a non-immunoglobulin domain comprises a structural domain oran epitope.

In a preferred embodiment, the Ig chain is joined to the non-Ig proteinvia a gene fusion. Preferably, the gene fusion is synthetic orrecombinant, and is generated using standard techniques of chemicalsynthesis or molecular biology. Typically, a mutation is introduced aspart of the gene fusion construct. Alternatively, a mutation may beintroduced subsequently, using known methods of mutagenesis (for exampleby exposing the gene fusion construct to irradiation, or chemical orbiological mutagenesis).

According to the invention, a junction region is the region of thefusion protein surrounding the junction point between the Ig and non-Igmoieties of the fusion protein. In a preferred embodiment, the junctionregion includes the C-terminal portion of the Ig moiety and theN-terminal portion of the non-Ig moiety. In one embodiment, the junctionregion also comprises a spacer or linker peptide inserted at thejunction point between the Ig and non-Ig moieties.

According to preferred embodiments of the invention, a mutation in theIg moiety is in the C-terminal portion of the Ig moiety, preferablywithin about 100 residues, more preferably within about 50 residues, orabout 25 residues, and even more preferably within about 10 residuesfrom the C-terminus of the Ig moiety.

According to preferred embodiments of the invention, a mutation in thenon-Ig moiety is in the N-terminal portion of the non-Ig moiety,preferably within about 100 residues, more preferably within about 50residues, or about 25 residues, and even more preferably within about 10residues from the N-terminus of the non-Ig moiety.

In preferred embodiments of the invention, a mutation is in theC-terminal region of the Ig moiety, but the mutation is not in part ofthe Ig protein that interacts with the Fc receptor (FcR) or FcRp.

An antibody fusion protein having a mutation according to the inventionhas an increased in vivo circulating half-life when compared to thecirculating half-life of a corresponding antibody fusion protein withoutthe mutation. The circulating half-life of an antibody fusion proteincan be measured by assaying the serum level of the fusion protein as afunction of time.

Experimental evidence indicates that the effects of preferred mutationsof the invention are not dependent on interactions with FcR or FcRp.First, preferred mutations that increase the circulating half-life of afusion protein do not affect regions of the antibody that, on the threedimensional structure, are part of the interaction surface that binds toFcR or to FcRp. Second, preferred mutations of the invention can causean improvement in serum half-life even when the interaction with FcR isremoved by use of an IgG-gamma4 chain and the interaction with FcRp isremoved by performing the pharmacokinetic study in a beta2-microglobulinmutant mouse in which FcRp is defective. Third, preferred mutations ofthe invention do not significantly affect the binding of Ig fusionproteins to FcR on J774 cells.

Site-directed mutagenesis analyses indicate that the surface of Fc thatinteracts with the Fc receptor is near the hinge region on the CH2domain. The Fc region's FcR interaction surface is very far, in threedimensions, from the C-terminus of Fc. Similar analyses indicate thatFcRp interacts with amino acid residues located at the interface betweenthe CH2 and CH3 domains.

FcRp binds its ligand with a much higher affinity at acidic pH (pH 6.0),than at neutral or slightly basic pH (pH 7.4). This is consistent withthe role of FcRp in protecting Fc containing molecules such asantibodies following their cellular internalization within endosomes.These cellular compartments become acidified after fusion with lysosomesand their protein constituents are degraded by acidic proteases. Bindingto membrane bound FcRp during this process prevents degradation of theantibody and allows it to be recycled to the outside of the cell (backinto the circulation) or across a cell layer (a process calledtranscytosis). This latter process allows IgG to pass through theneonatal intestinal mucosa following the ingestion of milk in the acidicenvironment of the gut.

The structure of the Fc/FcRp complex indicates that FcRp binds to theside of the Fc region, with contacts in both the CH2 and CH3 domains,and that the contacted region is not particularly close to theC-terminus of the Fc region. Thus, alteration of the very C-terminalregion of the Fc is not expected to alter the interaction with FcRp.

Not wishing to be bound by any particular theory, it is believed thatmutations in the fusion junction region that increase the circulatoryhalf life of a fusion protein according to the invention also reducecleavage of the fusion protein in a protease cleavage assay, asillustrated in Example 15. It is further believed that proteasedigestion may contribute to the disappearance of intact proteins formthe body, including fusion proteins. Thus, resistance to proteases maydirectly contribute to improved pharmacokinetics of proteins. It is alsofurther believed that protease digestion of non-denatured proteinsinvolves access by a protease to an exposed sequence in the correctconformation, as well as recognition of a specific sequence of aminoacids. Thus, mutations in the fusion junction that affect the generalconformation of a protein and thus affect accessibility of proteases totheir cleavage sites may contribute to protease resistance and toimproved pharmacokinetics. In addition, mutations that alter specificprotease recognition sequences may contribute to protease resistance andto improved pharmacokinetics.

A feature of mutations of the invention is that they can be combinedwith other mutations or substitutions in the antibody moiety tosynergistically modulate serum half-life or other properties of the Igmoiety. For example, one or more mutations of the invention thatincrease the circulating half-life of an antibody fusion protein can becombined with one or more mutations that affect the interaction betweenthe antibody fusion protein and FcR or FcRp.

In addition, the mutations of the invention can be used with a widevariety of antibody moieties and with a wide variety of non-Ig fusionpartners. The immunoglobulins include IgG, IgM, IgA, IgD, and IgE. Thenon-Ig fusion partners include cytokines, other secreted proteins,enzymes, or soluble fragments of transmembrane receptors, such asligand-binding domains.

According to the invention, an antibody-based fusion protein with anenhanced in vivo circulating half-life can be further enhanced bymodifying within the Fc portion itself. These may be residues includingor adjacent to Ile 253, His 310 or His 435 or other residues that caneffect the ionic environments of these residues when the protein isfolded in its 3-dimensional structure. The resulting proteins can betested for optimal binding at pH 6 and at pH 7.4-8 and those with highlevels of binding at pH 6 and low binding at pH 8 are selected for usein vivo. Such mutations can be usefully combined with the junctionmutations of the invention.

Methods and compositions of the invention are useful when coadministeredwith angiogenesis inhibitors such as those disclosed in PCT/US99/08335(WO 99/52562) or prostaglandin inhibitors such as those disclosed inPCT/US99/08376 (WO 99/53958). Methods and compositions of the inventioncan also be used in multiple cytokine protein complexes such as thosedisclosed in PCT/US00/21715. Methods and compositions of the inventionare also useful in combination with other mutations disclosed inPCT/US99/03966 (WO 99/43713) that increase the circulating half-life ofa fusion protein.

Non-limiting methods for synthesizing useful embodiments of theinvention are described in the Examples herein, as well as assays usefulfor testing pharmacokinetic activities, both in vitro and inpre-clinical in vivo animal models. The preferred gene constructencoding a chimeric chain includes, in 5′ to 3′ orientation, a DNAsegment which encodes at least a portion of an immunoglobulin and DNAwhich encodes a second, non-immunoglobulin protein. An alternativepreferred gene construct includes, in 5′ to 3′ orientation, a DNAsegment which encodes a second, non-immunoglobulin protein and DNA whichencodes at least a portion of an immunoglobulin. The fused gene isassembled in or inserted into an expression vector for transfection ofthe appropriate recipient cells where it is expressed.

The invention also provides methods for identifying mutations thatincrease the circulatory half-life of an antibody-based fusion protein.The methods comprise introducing one or more mutations in a regionspanning the junction between the Ig moiety and the non-Ig moiety of anantibody-based fusion protein. The circulating half-life of the mutatedfusion protein is assayed, preferably by monitoring its serum level invivo as a function of time.

In one embodiment of the invention, a mutation that increases thecirculatory half-life of an antibody-based fusion protein is a mutationthat reduces cleavage of the fusion protein in a protease cleavageassay, as discussed in Example 15. The mutation is preferably a mutationin a region spanning the junction between the Ig moiety and the non-Igmoiety of the fusion protein (for example, a mutation in the junctionregion discussed above). Alternatively, the mutation may be any mutationin the fusion protein that reduces protease cleavage and increases thecirculatory half life of the fusion protein, as described in Example 16.Accordingly, the invention provides methods for screening mutations inproteins in general, and preferably in an Ig-cytokine fusion protein, toidentify mutations that increase the circulatory half-life of the fusionprotein.

The invention is illustrated further by the following non-limitingexamples. The amino acid residue numbers used herein refer to the IgG1amino acid sequence. One of ordinary skill in the art will understandthat corresponding mutations in fusion proteins involving other Igproteins are useful to increase their circulating half-lives.

Accordingly, the teachings presented herein are applicable to other Igmolecules such as IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE.

EXAMPLES Example 1 Construction of Antibody-IL-2 Genes withSubstitutions of the Lys Codon at the Fusion Junction

The amino acid sequence at the junction of the antibody-IL-2 fusionprotein is SerProGlyLys-AlaProThr (SEQ ID NO: 1), in which theSerProGlyLys (SEQ ID No. 2) is the normal carboxy terminus of the heavychain of the antibody, and AlaProThr is the N-terminal sequence ofmature IL-2. In order to determine the effect alterations in the regionof the fusion junction on the pharmacokinetics of the fusion protein,substitutions or deletion of the residue were made by mutagenesis, asdescribed below.

The expression vector for immunocytokines was described in Gillies atal., (1998) J. Immunol. 160:6195-6203. In the human gamma-1 geneencoding the heavy chain, the XmaI restriction site located 280 bpupstream of the translation stop codon was destroyed by introducing asilent mutation (TCC to TCA). Another silent mutation (TCT to TCC) wasintroduced to the Ser codon three residues upstream of the C-terminallysine of the heavy chain to create the sequence TCC CCG GGT AAA (SEQ IDNo. 3), which contains a new XmaI site [Lo at al, (1998) ProteinEngineering 11:495-500]. The IL-2 cDNA was constructed by chemicalsynthesis and it contains a new and unique PvuII restriction site[Gillies at al., (1992) Proc. Natl. Acad. Sci. 89:1428-1432]. Both theXmaI and PvuII sites are unique in the expression vector, and theyfacilitated mutagenesis of the lysine codon which lies at the junctionof the CH3 and the IL-2 DNA.

Substitution or deletion of the Lys codon was achieved by replacing theXmaI-PvuII fragment in the immunocytokine expression vector with anoligonucleotide duplex encoding the desired mutation. In this case thevariable regions of the heavy and light chains were derived from thehumanized KS antibody, which recognized a human antigen called EpCAM(Epithelial cell adhesion molecule). The sequences of theoligonucleotide duplexes used in the present invention are listed below,where the codons in bold encode the desired mutations, and the sequencesin italics, CCCGG and CAG are the cohesive end of the XmaI site and theblunt end of the PvuII site, respectively. The oligonucleotide duplexwith 5′-hydroxyl ends were used in the ligation to the XmaI-PvuIIdigested expression vector. The use of oligonucleotides with 5′-hydroxylends eliminated self ligation of the oligonucleotide duplex.

1.) Lys to Ala Substitution

(SEQ ID NO: 4) 5′ CCG GGT GCA GCA CCT ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 5) 3′ CA CGT CGT GGA TGA AGT TCA AGA TGT TTC TTT TGTGTC 5′

2.) Lys to Arg Substitution

(SEQ ID NO: 6) 5′ CCG GGT AG G GCG CCA ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 7) 3′ CA TC C CGC GGT TGA AGT TCA AGA TGT TTC TTT TGTGTC 5′A NarI restriction site (GGCGCC) was also introduced by silent mutationto facilitate screening of recombinant clones.

3.) Deletion of Lys

(SEQ ID NO: 8) 5′ CCC GGT GCA CCT ACT TCA AGT TCT ACA AAG AAA ACA CAG 3′(SEQ ID NO: 9) 3′ CA CGT GGA TGA AGT TCA AGA TGT TTC TTT TGT GTC 5′

4.) Lys to Gly Substitution

(SEQ ID NO: 10) 5′ CCG GGT G GG GCC CCT ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 11) 3′ CA C CC CGG GGA TGA AGT TCA AGA TGT TTC TTTTGT GTC 5′An ApaI restriction site (GGGCCC) was also introduced by silent mutationto facilitate screening of recombinant clones.

5.) Lys to Leu Substitution

(SEQ ID NO: 12) 5′ CCG GGT CT G GCG CCA ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 13) 3′ CA GA C CGC GGT TGA AGT TCA AGA TGT TTC TTTTGT GTC 5′A NarI restriction site (GGCGCC) was also introduced by silent mutationto facilitate screening of recombinant clones.

6.) Lys to AlaAlaAla Substitution

(SEQ ID NO: 14) 5′ CCG GGT GCA GCA GCT GCC CCA ACT TCA AGT TCT ACA AAGAAA ACA CAG 3′ (SEQ ID NO: 15) 3′ CA CGT CGT CGA CGG GGT TGA AGT TCA AGATGT TTC TTT TGT GTC 5′

7.) Lys to Cys Substitution

(SEQ ID NO: 16) 5′ CCG GGT TGC GCA CCA ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 17) 3′ CA ACG CGT GGT TGA AGT TCA AGA TGT TTC TTT TGTGTC 5′A FspI restriction site (TGCGCA) was also introduced by silent mutationto facilitate screening of recombinant clones.

8.) Lys to Asp Substitution

(SEQ ID NO: 18) 5′ CCG GGT GAC GCA CCA ACT TCA AGT TCT ACA AAG AAA ACACAG 3′ (SEQ ID NO: 19) 3′ CA CTG CGT GGT TGA AGT TCA AGA TGT TTC TTT TGTGTC 5′

The recombinant gene constructs containing the various substitutions ordeletion of the Lys codon were confirmed by DNA sequencing.

Example 2 Construction of Antibody-IL-2 Genes Encoding Extra Amino AcidResidues at the Fusion Junction

It is common in the art to separate domains in fusion proteins withflexible linkers containing amino acid residues such as glycine andserine. The importance of the spacing between the CH3 and IL-2 wasstudied in the following mutagenesis experiments. Blunt endedoligonucleotide duplexes encoding different number of amino acidresidues were inserted into the SmaI endonuclease restriction site (samerecognition site as the XmaI mentioned above) of the huKS-IL-2expression vector by ligation; and the correct orientation of insertionwas confirmed by DNA sequencing. As discussed above, oligonucleotideduplexes with 5′-hydroxyl ends were used to eliminate self ligation.

9.) Lys to Cys Substitution with Linker Ligation

The following linker (oligonucleotide duplex) was inserted into the SmaIendonuclease restriction site of the huKS-IL-2 expression vector byligation. The sequence GCATGC encodes a SphI restriction site, whichfacilitated screening of recombinants containing the linker insertion.

5′ G GCA TGC GG 3′ 3′ C CGT ACG CC 5′

After linker ligation into the SmaI site (CCCGGG), the sequence at thefusion junction became

C CCG GCA TGC GGG GGT AAA (SEQ ID NO: 20) (linker sequence underlined)  Pro Ala Cys Gly Gly Lys (SEQ ID NO: 21)Therefore, the linker put a Cys residue at the original position of theLys residue, for a possible interchain disulphide bond formation. Theoriginal Lys residue was pushed back by 3 amino acid residues(AlaCysGly).

10.) A Linker Encoding 6 Amino Acid Residues

The following linker (oligonucleotide duplex) was inserted into the SmaIendonuclease restriction site of the huKS-IL-2 expression vector byligation. The sequence GGATCC encodes a BamHI restriction site, whichfacilitated screening of recombinants containing the linker insertion.

5′ G GGT TCA GGA TCC GGA GG 3′ (SEQ ID NO: 22) 3′ C CCA AGT CCT AGG CCTCC 5′ (SEQ ID NO: 23)After linker ligation into the SmaI site, the sequence at the fusionjunction became ProGlySerGlySerGlyGlyGlyLys (SEQ ID NO: 24), where thesix amino acid residues inserted were underlined.

11.) A Linker Encoding 11 Amino Acid Residues

The following linker (oligonucleotide duplex) was inserted into the SmaIendonuclease restriction site of the huKS-IL-2 expression vector byligation. The sequence GGATCC encodes a BamHI restriction site, whichfacilitated screening of recombinants containing the linker insertion.

(SEQ ID NO: 25) 5′ G GGT TCA GGC TCT GGA TCA GGG TCC GGA TCC GG 3′ (SEQID NO: 26) 3′ C CCA AGT CCG AGA CCT AGT CCC AGG CCT AGG CC 5′

After linker ligation into the SmaI site, the sequence at the fusionjunction became ProGlySerGlySerGlySerGlySerGly SerGlyGlyLys (SEQ ID NO:27), where the eleven amino acid residues inserted were underlined.

Example 3 Construction of Antibody-IL-2 Genes with Substitutions of thePro Codon at the Fusion Junction

The proline in the sequence ProGlyLys at the carboxyl terminus of CH3 ismutated to Ala, Leu or Gly, and other amino acids. This is accomplishedby replacing a 25 base-pair SapI-SmaI fragment of the KS-IL-2 expressionvector by an oligonucleotide duplex encoding the desired change. Each ofthe following oligonucleotide duplexes has a SapI cohesive end (3-baseoverhang) and a blunt end (for ligating to the SmaI end of therestriction fragment). The substitutions at the Pro codon are denoted inbold. These substitutions had no significant effect on thepharmacokinetics of the fusion protein, indicating that the structuralproperties of the Pro residue have no significant effect on thepharmacokinetics of the fusion protein

12.) Pro to Ala Substitution

(SEQ ID NO: 28) 5′ CG CAG AAG AGC CTC TCC CTG TCC GC 3′ (SEQ ID NO: 29)3′ TC TTC TCG GAG AGG GAC AGG CG 5′

13.) Pro to Leu Substitution

(SEQ ID NO: 30) 5′ CG CAG AAG AGC CTC TCC CTG TCC CT 3′ (SEQ ID NO: 31)3′ TC TTC TCG GAG AGG GAC AGG GA 5′

12.) Pro to Gly Substitution

(SEQ ID NO: 32) 5′ CG CAG AAG AGC CTC TCC CTG TCC GG 3′ (SEQ ID NO: 33)3′ TC TTC TCG GAG AGG GAC AGG CC 5′

Example 4 Construction of hu14.18-(Lys to Ala)-IL-2 DNA

In order to show that the effect of the Lys to Ala substitution on thepharmacokinetics of the antibody-IL-2 fusion protein was not limited tothe huKS antibody, we chose a different antibody, humanized 14.18(hu14.18), which recognized GD2, a ganglioside overexpressed on thesurface of many human tumor cells. The expression vector forhu14.18-(Lys to Ala)-IL-2 was constructed as described above.

Example 5 Construction of huKS-(Deleted Lys)-TNFα DNA

In order to show that the effect of the Lys residue on thepharmacokinetics of the antibody-IL-2 fusion protein was applicable toother cytokines, we chose a different cytokine, TNFα. The complete cDNAsequence of TNFα was published by Nedwin at al. in Nucleic Acids Res.(1985) 13:6361-6373, and the expression of an antibody-TNFα also hasbeen described by Gillies at al. in Bioconjugate Chem. (1993) 4:230-235.The fusion junction of the antibody-TNFα has the sequenceSerProGlyLys-ValArgSerSerSer (SEQ ID NO: 34), where Val is theN-terminal residue of the mature TNFα. In order to compare withhuKS-TNFα, DNA encoding huKS-(deleted Lys)-TNFα was constructed by anoverlapping PCR method [Daugherty at al., (1991) Nucleic Acids Res.19:2471-2476] with mutagenic primers encoding the deletion of the Lysresidue. The resultant expression vector for huKS-(deleted Lys)-TNFαtherefore encodes the peptide sequence SerProGly-ValArgSerSerSer (SEQ IDNO: 35) at the fusion junction. Additional modifications of this fusionprotein according to the new invention might include the removal of theArg residue in the amino terminal sequence of TNF to further reduce theoverall charge of the junction region.

Example 6 Construction of huKS-(EU)-(Lys to Ala)-IL-2 DNA

All the antibody-cytokine fusion proteins mentioned in the examplesabove were based on a certain allotype of the human IgG1 represented bythe myeloma H chain, KOL. In order to show that the effect of the Lys toAla substitution on the pharmacokinetics of the antibody-IL-2 fusionprotein was not limited to KOL, we chose a different IgG1 allotyperepresented by the myeloma H chain, EU. The EU allotype differs from theKOL allotype in three amino acid residues in the constant regions. TheEU allotype contains Lys-229 at the end of CH1, and Asp-356 and Leu-358at the beginning of CH3. The KOL allotype contains Arg-229, Glu-356 andMet-358 at the corresponding positions. The DNA encoding the EU allotypewas obtained by mutagenesis of the KOL DNA using the overlapping PCRmethod. The resultant EU DNA was then used to replace the correspondingfragment of the KOL DNA to generate the expression vector for producinghuKS-(EU)-(Lys to Ala)-IL-2.

Example 7 Transfection of Cells and Expression of Proteins

For transient transfection, the plasmid was introduced into Baby HamsterKidney (BHK) cells by lipofection using Lipofectamine Plus (LifeTechnologies, Gaithersburg, Md.) according to supplier's protocol.

In order to obtain stably transfected clones, plasmid DNA was introducedinto the mouse myeloma NS/0 cells by electroporation. NS/0 cells weregrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum, 2 mM glutamine and penicillin/streptomycin. About 5×10⁶cells were washed once with PBS and resuspended in 0.5 ml PBS. Ten μg oflinearized plasmid DNA were then incubated with the cells in a GenePulser Cuvette (0.4 cm electrode gap, BioRad) on ice for 10 min.Electroporation was performed using a Gene Pulser (BioRad, Hercules,Calif.) with settings at 0.25 V and 500 μF. Cells were allowed torecover for 10 min. on ice, after which they were resuspended in growthmedium and then plated onto two 96 well plates. Stably transfectedclones were selected by growth in the presence of 100 nM methotrexate(MTX), which was introduced two days post-transfection. The cells werefed every 3 days for two to three more times, and MTX-resistant clonesappeared in 2 to 3 weeks. Supernatants from clones were assayed byanti-Fc ELISA to identify high producers. High producing clones wereisolated and propagated in growth medium containing 100 nM MTX.

For routine characterization by gel electrophoresis, antibody-cytokinefusion proteins in the conditioned media were captured on Protein ASepharose (Repligen, Cambridge, Mass.) and then eluted by boiling in theprotein sample buffer with or without 2-mercaptoethanol. Afterelectrophoresis on an SDS gel, the protein bands were visualized byCoomassie staining. The antibody heavy chain-IL-2 and the light chainhad apparent MW of about 67 and 28 kD respectively, on SDS-PAGE.

For purification, the fusion proteins bound on Protein A Sepharose wereeluted in a sodium phosphate buffer (100 mM NaH₂PO₄, pH 3, and 150 mMNaCl). The eluate was then immediately neutralized with 0.1N NaOH.

Example 8 ELISA Procedures

ELISAs were used to determine the concentrations of protein products inthe supernatants of MTX-resistant clones and other test samples. Theanti-huFc ELISA consists of a capturing step using goat anti-human IgG(against both heavy and light chains) and a detection step using thehorseradish peroxidase-conjugated F(ab′)₂ fragment of goat anti-humanIgG, Fc fragment specific. Therefore, the anti-huFc ELISA measures humanIgG, either as an antibody by itself or as a cytokine fusion protein. Todetermine the concentration of the intact antibody-IL-2 fusion protein,an IL-2-detection ELISA was used. It consists of the same capturing stepusing goat anti-human IgG (against both heavy and light chains), but thedetection step uses a detection antibody directed against IL-2. In someexperiments, EPCAM was used instead of a capture antibody to detectKS-IL-2 fusion proteins, since the KS antibody recognizes EPCAM. In someexperiments, a commercial human IL-2 ELISA detection kit was used (R&DSystems). All the different ELISA procedures involving IL-2 detectionantibodies gave similar results. However, as can be seen from acomparison of FIG. 1A and FIG. 1B, there is a progressive loss ofIL-2-immunoreactive material compared to human Fc immunoreactivematerial in later pharmacokinetic time points. This effect is mostpronounced for fusion proteins that have the poorest pharmacokineticproperties.

The anti-huFc ELISA is described in detail below.

A. Coating Plates.

ELISA plates were coated with AffiniPure Goat anti-Human IgG (H+L)(Jackson Immuno Research Laboratories, West Grove, Pa.) at 5 μg/mL inPBS, and 100 μL/well in 96-well plates (Nunc-Immuno plate Maxisorp).Coated plates were covered and incubated at 4° C. overnight. Plates werethen washed 4 times with 0.05% Tween (Tween 20) in PBS, and blocked with1% BSA/1% goat serum in PBS, 200 μL/well. After incubation with theblocking buffer at 37° C. for 2 hrs, the plates were washed 4 times with0.05% Tween in PBS and tapped dry on paper towels.

B. Incubation with Test Samples and Secondary Antibody

Test samples were diluted to the proper concentrations in sample buffer,which contains 1% BSA/1% goat serum/0.05% Tween in PBS. A standard curvewas prepared with a chimeric antibody (with a human Fc), theconcentration of which was known. To prepare a standard curve, serialdilutions are made in the sample buffer to give a standard curve rangingfrom 125 ng/mL to 3.9 ng/mL. The diluted samples and standards wereadded to the plate, 100 μL/well and the plate was incubated at 37° C.for 2 hr. After incubation, the plate was washed 8 times with 0.05%Tween in PBS. To each well was then added 100 μL of the secondaryantibody, the horseradish peroxidase-conjugated AffiniPure F(ab′)₂fragment goat anti-human IgG, Fc fragment specific (Jackson ImmunoResearch), diluted around 1:120,000 in the sample buffer. The exactdilution of the secondary antibody has to be determined for each lot ofthe HRP-conjugated anti-human IgG. After incubation at 37° C. for 2 hr,the plate was washed 8 times with 0.05% Tween in PBS.

C. Development

The substrate solution was added to the plate at 100 μL/well. Thesubstrate solution was prepared by dissolving 30 mg of OPD(o-phenylenediamine dihydrochloride, 1 tablet) into 15 mL of 0.025 MCitric acid/0.05 M Na₂HPO₄ buffer, pH 5, which contained 0.03% offreshly added H₂O₂. The color was allowed to develop for 30 min. at roomtemperature in the dark. The developing time is subject to change,depending on lot to lot variability of the coated plates, the secondaryantibody, etc. Watch the color development in the standard curve todetermine when to stop the reaction. The reaction was stopped by adding4N H₂SO₄, 100 μL/well. The plate was read by a plate reader, which wasset at both 490 and 650 nm and programmed to subtract the background ODat 650 nm from the OD at 490 nm.

Example 9 Pharmacokinetic Behavior of Antibody-Cytokine Fusion ProteinsCarrying Alterations at the Fusion Junction

The fusion proteins were tested for their pharmacokinetic behaviorfollowing intravenous injection into Balb/c mice. Blood was collectedfrom mice by retro-orbital bleeding and stored at 4° C. in Eppendorfmicro-centrifuge tubes. In some cases, two different ELISA methods wereused to measure both the amount of human antibody and the amount ofsecond, fused non-Ig protein remaining in the blood at various timepoints. Alternatively, the presence of the non-Ig moiety was inferred byWestern blot analysis of pharmacokinetic time points.

Using the techniques described in the preceding examples, theKS(gamma1)-IL-2 fusion mutant proteins were injected into mice, and theeffect on serum half-life was determined. Some of the results are shownin FIG. 1 and FIG. 2. In addition, the effect of deletion of theantibody heavy chain's C-terminal lysine was examined in anIgG(gamma1)-IL-2 fusion in which the antibody had a different bindingspecificity. The pharmacokinetic properties of a 14.18(Lys→Ala)-IL-2were superior to 14.18-IL-2 to an extent that was similar to theimprovement of KS(Lys→Ala)-IL-2 as compared to KS-IL-2.

For antibody-IL-2 fusions, the ranking of the effect of mutationsaffecting the C-terminal lysine of the heavy chain on thepharmacokinetic properties was (from best to worst):Lys→Leu˜Lys→Ala˜Lys→Ala₃>Lys→(deleted)>Lys→Asp˜Lys→Gly>Lys→(nochange)˜Lys→Cys>Lys→Arg.

The pharmacokinetic properties of KS(Lys→deleted)-TNFalpha weresignificantly improved as compared to KS-TNFalpha (FIG. 3). Thepharmacokinetic profile of the KS-TNFalpha fusion protein was unusual inthat, when the levels of human antibody are measured by Fc ELISA, therewas a sharp drop in the level of detected protein within the first 30minutes, followed by a slow increase in the level of human Fc-reactivematerial. This effect was highly reproducible.

When pharmacokinetic samples were analyzed by Western blotting, it wasfound that human Fc-cross-reactive material was in the form of intactantibody; the TNF moiety had been cleaved off and lost. However, similaranalysis of the KS-TNFalpha fusion protein carrying a deletion of theC-terminal lysine indicated that this protein survived primarily in anintact form, with TNF still present.

In addition, a KS-TNFalpha fusion protein was expressed in which thefirst eight amino acids of the mature TNFalpha sequence were deleted.The pharmacokinetic properties of the deleted KS-TNFalpha fusion proteinwere superior to corresponding proteins having the entire mature TNFsequence. This is likely due to removal of the charged Arg residue atthe +2 position of the mature TNF which increases the hydrophobicity ofthe junctional region.

Changing the heavy chain constant regions of KS(Lys→Ala)-IL-2 andKS-IL-2 from KOL to EU had no effect on the pharmacokinetic propertiesof either protein.

Taken together, these results indicate that mutation of the junctioncaused a significant improvement of the pharmacokinetic properties of Igfusion proteins. The effect was seen with diverse antibodies, anddiverse non-Ig proteins fused to an Ig moiety.

Example 10 Combining Mutations at the Fusion Junction with a Change inIg Type from Gamma1 to Gamma4 Leads to a Synergistic Enhancement ofSerum Half-Life that is Independent of FcRp Function

The human gamma4 Fc region binds poorly to Fc receptors. As a result,fusion proteins that comprise a gamma4 Fc region generally have asuperior pharmacokinetic properties as compared to fusion proteinshaving the gamma1 chain. To address whether junction mutations affectpharmacokinetics through an effect on an Fc receptor interaction, anFcRp interaction, or both, the pharmacokinetic properties of gamma1- andgamma4-containing fusion proteins with or without junction mutationswere examined in mice that were either normal or defective in FcRp. Theresults of these pharmacokinetic experiments are shown in FIG. 2.

FIG. 2 shows the pharmacokinetic behavior of a KS(gamma1)-IL-2 fusionprotein, a KS(gamma4)-IL-2 fusion protein, a KS(gamma1)(Lys-to-Ala)-IL-2fusion protein, and a KS(gamma4)(Lys-to-Ala)-IL-2 fusion protein. Normalmice and mutant mice defective in beta2 microglobulin were examined.

These data indicated that, in a normal mouse, the pharmacokinetics of anIgG-gamma1 antibody-IL-2 fusion protein were improved by introducing aLys-to-Ala mutation at the C-terminus of the antibody moiety. Similarly,the pharmacokinetics of an IgG-gamma4 antibody-IL-2 fusion protein wereimproved by introducing a Lys-to-Ala mutation at the C-terminus of theantibody moiety. These data indicate that a junction mutation canimprove the pharmacokinetic properties of a fusion protein that alreadyhas improved pharmacokinetics as a result of reduced Fc receptorbinding.

FIG. 2 also shows the pharmacokinetic properties of the same proteinswhen injected into mutant mice lacking the beta2-microglobulin protein,which is an essential subunit of FcRp (Junghans and Anderson, Proc. NatAcad. Sci. (1996) 93:5512-5516). Thus, these mutant mice are defectivein FcRp activity. As a result, the catabolism of antibodies is about10-fold faster in such mutant mice than in normal mice.

The data of FIG. 2 indicated that the KS (gamma1) antibody, a KS(gamma1)-IL-2 fusion protein, a KS (gamma4)-IL-2 fusion protein, a KS(gamma1)(Lys-to-Ala)-IL-2 fusion protein, and a KS(gamma4)(Lys-to-Ala)-IL-2 fusion protein all were catabolized morerapidly in the beta2-microglobulin mutant mice than in wild-type mice.However, the relative order of serum half-lives is the same for theseproteins in both mouse strains: the unfused antibody has the bestpharmacokinetics, followed by the KS(gamma4)(Lys-to-Ala)-IL-2 fusionprotein, the KS(gamma1)(Lys-to-Ala)-IL-2 fusion protein, theKS(gamma4)-IL-2 fusion protein, with the KS(gamma1)-IL-2 fusion proteinhaving the worst pharmacokinetic properties. If a junction mutation hadits effect exclusively by changing the interaction of a fusion proteinwith FcRp, then in the absence of FcRp function, the junction mutationshould have no effect on pharmacokinetics.

Example 11 Mutation of the Junction Region in an Intact Antibody has NoEffect on Serum Half Life

A mutation in a gene encoding the heavy chain of the intact, unfused KSantibody is engineered to change the C-terminal lysine to an alanine.The wild-type and mutant forms of KS are expressed and purified by themethods described above, and the pharmacokinetic properties arecompared. The pharmacokinetic behaviors of the wild-type and mutantantibodies are found to be indistinguishable.

Example 12 Binding to Fc Receptor by Antibody Fusion Proteins with orwithout Mutations at the Fusion Junction

Using a standard procedure, the binding of KS-IL-2 and KS(K-A)-IL-2 toFc receptors was examined. No effect of the mutation was found. Fusionproteins were expressed and purified as described above, and were testedfor their ability to bind to fixed J774 cells, which express the Fcreceptor. Results are shown in FIG. 4.

Example 13 Treatment of Colon Carcinoma in a Mammal with anAntibody-Cytokine Fusion Protein Containing a Junction Mutation

To test whether a cytokine-antibody fusion protein with a junctionmutation would be advantageous in treatment of colon carcinoma in amammal, the following experiments were performed. CT26 is a coloncarcinoma cell line derived from Balb/C mice. By standard geneticengineering techniques, this cell line was engineered to express thehuman epithelial cell adhesion molecule (EpCAM), which is the antigenrecognized by the KS antibody; these cells are termed CT26/EpCAM cells(Gillies at al. Journal of Immunology (1998) 160:6195-6203).

Balb/C mice were subcutaneously inoculated with 2×10⁶ CT26/EpCAM cells.When tumors reached a volume of about 50-200 cubic millimeters, micewere randomized into three groups of 7 mice for further study. Beginningat day 0, tumor-bearing mice were treated with PBS, about 10 microgramsof KS-IL2 with an IgG1 heavy chain (KS-IL2gamma1), or about 10micrograms of KS-IL2 with an IgG1 heavy chain and the Lys to Alamutation described in the previous examples (KS-IL2gamma1 [Lys to Ala]).Mice were injected intravenously, once per day for five days. Tumorsizes were measured with calipers.

The results of one such experiment are shown in FIG. 5. In thisexperiment, KS-IL2gamma1 caused a significant decrease in the volume ofmany, but not all tumors. In six of the seven KS-IL2gamma1-treatedanimals, tumors were still measurable on day 21. However, in theKS-IL2gamma1 (Lys to Ala)-treated animals, the tumors shrank, so that byday 21, the tumors in all seven animals were unmeasurable, and by day16, only two of seven mice had measurable tumors. In FIG. 5, blackdiamonds indicate average tumor volumes in mice that were injected withPBS as controls on days 0, 1, 2, 3, and 4. Filled circles indicateaverage tumor volumes in mice treated with 10 micrograms of KS-IL2gamma1. Intravenous injections were performed. The x-axis indicates thenumber of days elapsed following the first injection; the y-axisindicates the average tumor volume in cubic milliliters.

Example 14 Inhibition of Metastasis in a Mammal Treated with anAntibody-Cytokine Fusion Protein Containing a Junction Mutation

To test whether an antibody-cytokine fusion protein could inhibitmetastatic growth of tumor cells, the following experiments wereperformed. Lewis Lung Carcinoma (LLC) is a lung carcinoma cell linederived from C57/B16 mice. By standard genetic engineering techniques,this cell line was engineered to express the human epithelial celladhesion molecule (EpCAM), which is the antigen recognized by the KSantibody; these cells are termed LLC/EpCAM cells.

C57/B16 mice were intravenously injected with 1×10⁶ LLC/EpCAM cells.After five days, mice were randomized into three groups of 6 mice andtreated with either PBS, about 20 micrograms of KS-IL2, or about 20micrograms of KS-Ala-IL2 (KS-IL2 with a Lys to Ala change at theC-terminus of the Ig moiety). Metastases were quantitated on day 24. Asindicated in the table below, the PBS-treated group had large numbers ofmetastases into the lungs. Animals treated with KS-γ1-IL2 had asignificantly reduced number of metastases. However, animals treatedwith KS-γ1-ala-IL2 had even fewer metastases than animals treated withKS-γ1-IL2, and in one animal, no metastases at all were detected.

Treatment Group Number of Metastases Lung Wt. (g)PBS >250, >250, >250, >250, 0.92 +/− 0.14 >250, >250 KS-γ1-IL2 62, 37,18, 17, 11, 9 0.27 +/− 0.04 KS-γ1-ala-IL2 4, 4, 3, 3, 1, 0 0.25 +/− 0.02

Taken together, Examples 13 and 14 illustrate that antibody-cytokinefusion proteins can inhibit establishment of metastases as well asgrowth of tumor cells at the primary site. In addition, the resultsindicate that antibody-cytokine fusion proteins can inhibit diseaseresulting from a variety of different tumor types, such as colon cancerand lung cancer. Furthermore, antibody-cytokine fusion proteins with atleast one amino acid change in the linker region in accordance with theinvention are more effective at inhibiting metastases and tumor growththat antibody-cytokine fusion proteins with no amino acid change in thelinker region.

Example 15 Assay of Antibody Fusion Proteins with Junction Mutations forResistance to Proteases

To address whether antibody-cytokine fusion proteins with junctionmutations were more or less sensitive to protease digestion, purifiedKS-IL2 and KS-Ala-IL2 were treated with various proteases for varioustimes, and the resulting products were analyzed by SDS-PAGE.

In one experiment, 4 micrograms of KS-IL2 and KS-Ala-IL2 were treatedwith 0.1 mU or 0.4 mU of Cathepsin D (Enzyme Systems, Livermore, Calif.)for about 16 hours at 37 degrees C. and analyzed by SDS-PAGE. Bufferconditions were used according to the manufacturer's instructions. WhenKS-IL2 was treated with 0.4 mU of Cathepsin D, about 50% of the KS-IL2heavy chain was converted to various lower molecular weight forms. Thedominant digestion product had a molecular weight slightly less thanthat of KS-IL2 heavy chain, but much larger than the KS heavy chain.This result indicates that most of the cleavage by Cathepsin D was nottaking place at the heavy chain-IL2 junction.

In contrast, when KS-Ala-IL2 was incubated with 0.4 mU of Cathepsin Dunder the same conditions, the extent of cleavage by Cathepsin D wasmuch less, and a band with the molecular weight of the major KS-IL2degradation product was essentially undetectable.

In a second experiment, 4 micrograms of KS-IL2 and KS-Ala-IL2 weretreated with 25 mU or 50 mU of Cathepsin L (Enzyme Systems, Livermore,Calif.) for about 16 hours at 37 degrees C. and analyzed by SDS-PAGE.Buffer conditions were used according to the manufacturer'sinstructions. When KS-IL2 was treated with 50 mU of Cathepsin L, almostall of the KS-IL2 heavy chain was converted to various lower molecularweight forms. The dominant digestion product had a molecular weightabout the same as the KS heavy chain. This result indicates that much ofthe cleavage by Cathepsin L was taking place near or at the heavychain-IL2 junction.

In contrast, when KS-Ala-IL2 was incubated with 50 mU of Cathepsin Lunder the same conditions, the extent of cleavage by Cathepsin L wasmuch less, and a band with the molecular weight of the major KS-IL2degradation product was still the major molecular weight speciesobserved.

In a third experiment, 4 micrograms of KS-IL2 and KS-Ala-IL2 weretreated with 0.04 mU, 0.1 mU or 0.2 mU of plasmin (Sigma, St. Louis,Minn.) for about 16 hours at 37 degrees C. and analyzed by SDS-PAGE.Buffer conditions were used according to the manufacturer'sinstructions. When KS-IL2 was treated with 0.04 mU of plasmin, about ¾of the KS-IL2 heavy chain was converted to a lower molecular weight formwith an apparent molecular weight about 30 amino acids greater than thatof the KS heavy chain. When KS-IL2 was treated with 0.2 mU of plasmin,essentially all of the KS-IL2 heavy chain was converted to a lowermolecular weight form with an apparent molecular weight about 30 aminoacids greater than that of the KS heavy chain. These results indicatethat the cleavage of KS-IL2 by plasmin was taking place close to, butnot at the heavy chain-IL2 junction.

In contrast, when KS-Ala-IL2 was incubated with 0.04 mU of plasmin underthe same conditions, the extent of cleavage by plasmin was barelydetectable. When KS-Ala-IL2 was incubated with 0.2 mU of plasmin, someuncleaved product was detected. In addition when KS-Ala-IL2 was cleavedwith plasmin, a species with a molecular size about 90 amino acidsgreater than the KS-IL2 heavy chain accumulated to a significant extent;in the KS-IL2 digestions by plasmin, this +90 species was probablyrapidly cleaved to the lower molecular weight +30 species, and thusfailed to accumulate. Nonetheless, the Lys-to-Ala mutation caused asignificant stabilization of intact KS-IL2 in the presence of plasmin.In each case, the antibody light chain was uncleaved under theconditions used.

Taken together, these results indicated that the Lys-to-Ala mutationcaused a general resistance to protease cleavage, even to cleavages thatdo not take place at the site of the mutation. Without wishing to bebound by any particular theory, the Lys-to-Ala mutation may cause theIL-2 moiety of KS to become more resistant to proteases. Proteases mayplay an important role in the pharmacokinetic properties of antibodyfusion proteins. For example, when antibody fusion proteins are taken upby cells bearing an Fc receptor and transported into the early endosome,it may be that the antibody moiety is resistant to the proteolyticconditions used, but that the fusion partner moiety is more sensitive,resulting in partial or complete digestion of the antibody fusionprotein.

Example 16 Use of Protease Digestion to Evaluate Mutations in AntibodyFusion Proteins

This example provides a general method for improving the pharmacokineticproperties of a protein. A protein is tested for its pharmacokineticproperties and also its sensitivity to proteases. Variant proteins aregenerated and tested for greater resistance to proteolysis. Thosevariants with enhanced resistance to proteolysis are then tested fortheir pharmacokinetic properties. It is found that the proportion ofproteolysis-resistant proteins with improved pharmacokinetic propertiesis greater than for the population of variant proteins as a whole. Somevariant proteins with improved pharmacokinetic properties have one ormore amino acid substitutions that do not cause a profound change in theprotein structure that can be inferred by inspection of the encodingsequence, such as introduction of an N-linked glycosylation site.

Variant proteins are generated by, for example, mutagenesis of anexpression construct and isolation of clones expressing individualvariant proteins. Any of a variety of mutagenesis techniques is used,including site-directed mutagenesis, random mutagenesis,PCR-mutagenesis, and mutagenesis techniques that generate hybridsequences from related genes.

It is useful to use intracellular proteases, such as endosomalproteases, for these assays. Without wishing to be bound by anyparticular theory, it is believed that the pharmacokinetics of certainproteins, particularly proteins that are not removed by renalfiltration, is determined by proteolysis that occurs upon endocytosis.

It is also useful to use extracellular proteases, such as trypsin,chymotrypsin, plasmin, other digestive protease, other serum proteasessuch as clotting factors, and tissue-specific proteases. For example,tumor-specific proteases are used to test variant proteins and identifythose variants that have improved pharmacokinetic properties andstability within the tumor microenvironment. In another example,proteins that are to be orally delivered are tested for their resistanceto enzymes present in the gastro-intestinal tract, such as trypsin andchymotrypsin. It is found that variant proteins with enhanced resistanceto gastro-intestinal enzymes have improved pharmacokinetic properties,such as a greater AUC (Area Under the Curve).

For example, an expression construct encoding a fusion proteincontaining part or all of an antibody is mutagenized. Clones aregenerated, the corresponding proteins are expressed, and the proteinsare tested, either individually or in small pools, for relativesensitivity to proteases. Variant antibody fusion proteins with enhancedresistance to proteases are then tested for their pharmacokineticproperties, and a significant number of the protease-resistant antibodyfusion protein variants have improved pharmacokinetic properties. Thenucleic acids encoding the improved variant fusion proteins aresequenced, and some improved variants are found to contain mutations atsites other than the fusion protein junction that cause the phenotype ofenhanced resistance to proteolysis and improved pharmacokinetics.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

Each of the patent documents and scientific publications disclosedherein is incorporated by reference into this application in itsentirety.

1-47. (canceled)
 48. A method for increasing the circulating half-lifeof an antibody-based fusion protein having an immunoglobulin (Ig) chainlinked to a non-Ig protein via a junction point, the method comprisingexpressing the antibody-based fusion protein as an N-terminal Ig chainlinked to a C-terminal non-Ig protein, the Ig chain comprising an IgG2,IgG3, IgG4, IgA, IgM, IgD, or IgE constant domain and an amino acidsubstitution introducing a hydrophobic or non-polar amino acid within 10amino acids from the C-terminus of the Ig chain.
 49. The method of claim48, wherein the constant domain comprises at least one of a CH1, CH2, orCH3 domain.
 50. The method of claim 48, wherein the constant domaincomprises at least one of a CH2 or CH3 domain.
 51. The method of claim48, wherein said amino acid substitution increases the hydrophobicity ofsaid antibody-based fusion protein.
 52. The method of claim 48, whereinsaid substitution replaces a charged amino acid with a hydrophobic ornonpolar amino acid.
 53. The method of claim 48, wherein saidsubstitution changes the C-terminal amino acid of the Ig chain.
 54. Themethod of claim 53, wherein the Ig chain comprises the CH2 domain of theIgG2 constant region and a C-terminal lysine is substituted with anon-polar or hydrophobic amino acid.
 55. The method of claim 54, whereinthe C-terminal lysine is substituted with an alanine.
 56. The method ofclaim 54, wherein the non-Ig protein is a cytokine.
 57. The method ofclaim 48, wherein the Ig chain comprises part of an Ig heavy chain. 58.The method of claim 57, wherein said part of an Ig heavy chain furtherhas binding affinity for an immunoglobulin protection receptor.
 59. Themethod of claim 57, wherein said Ig chain has substantially reducedbinding affinity for a Fc receptor selected from the group consisting ofFcγRI, FcγII and FcγRIII, when compared to the binding affinity of anunsubstituted Ig chain for said Fc receptor.
 60. The method of claim 48,wherein the Ig chain comprises at least the CH2 domain of an IgG2 or anIgG4 constant region.
 61. The method of claim 48, further comprising alinker between said Ig chain and said non-Ig protein.
 62. The method ofclaim 48, wherein said hydrophobic or non-polar amino acid is selectedfrom the group consisting of Leu, Trp, Ala, and Gly.
 63. The method ofclaim 62, wherein said hydrophobic or non-polar amino acid is selectedfrom the group consisting of Trp, Ala, and Gly.
 64. The method of claim48, wherein said non-Ig protein is a secreted protein.
 65. The method ofclaim 64, wherein said non-Ig protein is a mature form of said secretedprotein.
 66. The method of claim 48, wherein said non-Ig protein isinterleukin-2.
 67. A method for increasing the circulating half-life ofan antibody-based fusion protein having an immunoglobulin (Ig) chainlinked to a non-Ig protein via a junction point, the method comprisingexpressing the antibody-based fusion protein as an N-terminal Ig chainlinked to a C-terminal non-Ig protein, the Ig chain comprising: at leastone of a CH2 and CH3 domain; and an amino acid sequence that isnon-natural within 10 amino acids from its C-terminus, the non-naturalamino acid sequence comprising an amino acid substitution introducing ahydrophobic or non-polar amino acid.
 68. The method of claim 67, whereinthe Ig chain is an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE chain.69. The method of claim 67, wherein said amino acid substitutionincreases the hydrophobicity of said antibody-based fusion protein. 70.The method of claim 67, wherein said amino acid substitution replaces acharged amino acid with a hydrophobic or non-polar amino acid.
 71. Themethod of claim 67, wherein said substitution changes the C-terminalamino acid of the Ig chain.
 72. The method of claim 71, wherein the Igchain comprises the CH2 domain of the IgG2 constant region and aC-terminal lysine is substituted with a nonpolar or hydrophobic aminoacid.
 73. The method of claim 72, wherein the C-terminal lysine issubstituted with an alanine.
 74. The method of claim 72, wherein thenon-Ig protein is a cytokine.
 75. The method of claim 67, wherein the Igchain comprises at least the CH2 domain of an IgG2 or an IgG4 constantregion.
 76. The method of claim 67, further comprising a linker betweensaid Ig chain and said non-Ig protein.
 77. The method of claim 67,wherein said hydrophobic or non-polar amino acid is selected from thegroup consisting of Leu, Trp, Ala, and Gly.
 78. The method of claim 77,wherein said hydrophobic or non-polar amino acid is selected from thegroup consisting of Trp, Ala, and Gly.
 79. A method for increasing thecirculating half-life of an antibody-based fusion protein having animmunoglobulin (Ig) chain linked to a non-Ig protein via a junctionpoint, the method comprising expressing the antibody-based fusionprotein as an N-terminal immunoglobulin (Ig) chain linked to aC-terminal non-Ig protein, the Ig chain comprising an amino acidsubstitution within 10 amino acids from the C-terminus, the substitutionreplacing a charged amino acid with a hydrophobic or non-polar aminoacid.
 80. The method of claim 79, wherein said hydrophobic or non-polaramino acid is selected from the group consisting of Leu, Trp, Ala, andGly.
 81. The method of claim 80, wherein said hydrophobic or non-polaramino acid is selected from the group consisting of Trp, Ala, and Gly.82. A method for increasing the circulating half-life of anantibody-based fusion protein having an immunoglobulin (Ig) chain linkedto a non-Ig protein via a junction point, the method comprisingexpressing the antibody-based fusion protein as an N-terminalimmunoglobulin (Ig) chain linked to a C-terminal non-Ig protein, theN-terminal Ig chain comprising an amino acid substitution within 10amino acids from the C-terminus of the Ig chain, the substitutionintroducing a hydrophobic or non-polar amino acid selected from thegroup consisting of Ala, Gly and Trp.
 83. A method for increasing thecirculating half-life of an antibody-based fusion protein having animmunoglobulin (Ig) chain linked to a non-Ig protein via a junctionpoint, the method comprising expressing the antibody-based fusionprotein as an N-terminal Ig chain linked to a C-terminal non-Ig protein,the C-terminal non-Ig protein comprising an amino acid substitutionintroducing a hydrophobic or non-polar amino acid within 10 amino acidsof the N-terminus of the C-terminal non-Ig protein.
 84. The method ofclaim 83, wherein said amino acid substitution increases thehydrophobicity of said antibody-based fusion protein.
 85. The method ofclaim 83, wherein the Ig chain comprises (i) a deletion of a chargedamino acid within 10 amino acids of the C-terminus of the Ig chain, or(ii) an amino acid alteration introducing a hydrophobic or non-polaramino acid within 10 amino acids of the C-terminus of the Ig chain. 86.The method of claim 83, wherein the hydrophobic or non-polar amino acidis Ala.
 87. The method of claim 83, further comprising a linker betweensaid Ig chain and said non-Ig protein.
 88. The method of claim 83,wherein said hydrophobic or non-polar amino acid is selected from thegroup consisting of Leu, Trp, Ala, and Gly.
 89. A method for increasingthe circulating half-life of an antibody-based fusion protein having animmunoglobulin (Ig) chain linked to a non-Ig protein via a junctionpoint, the method comprising expressing the antibody-based fusionprotein as an N-terminal immunoglobulin (Ig) chain linked to aC-terminal non-Ig protein, the Ig chain comprising at least one of a CH2and a CH3 domain, and the C-terminal non-Ig protein comprising an aminoacid alteration within 10 amino acids of the N-terminus of theC-terminal non-Ig protein, the alteration introducing an amino acidselected from the group consisting of Leu and Trp.